Window of Opportunity

mtcPTM is an online repository of human and mouse phosphosites in which data are hierarchically organized to preserve biologically relevant experimental information, thus allowing straightforward comparisons of phosphorylation patterns found under different conditions. The database also contains the largest available collection of atomic models of phosphorylatable proteins. Detailed analysis of this structural dataset reveals that phosphorylation sites are found in a heterogeneous range of structural and sequence contexts. mtcPTM is available on the web http://www.mitocheck.org/cgi-bin/mtcPTM/search. Rationale In recent years, several sequencing projects have revealed the complete transcriptomes and proteomes for a number of organisms, including human [1,2]. The current challenge is to place this information within the dynamic context of the cell in order to elucidate how individual molecules interact to achieve the complex behavior of cellular processes, which translates into the ability of living organisms to adapt and thrive in a myriad of environments and conditions. Thus, much effort has been invested in identifying, for example, the transcription patterns of genes and the interacting partners of proteins in order to determine the connections that establish the intricate cellular pathways [3,4]. To understand these networks fully, however, we must also comprehend how their connections are regulated when the states of individual components are altered, for example by means of post-translational modifications (PTMs). It is therefore crucial to identify which proteins can be modified as well as the effect and lifetime of the PTMs. Among PTMs, reversible protein phosphorylation is known to play a key role in regulating a variety of processes in eukaryotes, from the cell division cycle to neuronal plasticity [5,6]. The most commonly observed phosphorylations affect serine, threonine, and tyrosine residues [7,8], although phosphorylation of histidines and aspartates has also been reported (for review [9]). Protein phosphorylation is catalyzed by enzymes called protein kinases, which are usually specific for either tyrosine or serine/threonine, with few of them being able to modify all three residues indistinguishably [10-12]. The human genome encodes 518 protein kinases [13,14], and recent estimates suggest that around one-third of cellular proteins could undergo phosphorylation [15]. Despite the progress made during the past few decades, our knowledge about regulation of protein function by phosphorylation and the basis of kinase specificity remains incomplete, mainly because of lack of data. High-throughput proteomic approaches are expected to help fill this gap because they can identify large amounts of in vivo modified peptides (for review [16,17]). Published: 23 May 2007 Genome Biology 2007, 8:R90 (doi:10.1186/gb-2007-8-5-r90) Received: 3 January 2007 Revised: 3 April 2007 Accepted: 23 May 2007 The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2007/8/5/R90 Genome Biology 2007, 8:R90 R90.2 Genome Biology 2007, Volume 8, Issue 5, Article R90 Jiménez et al. http://genomebiology.com/2007/8/5/R90 Protein kinases catalyze the formation of a covalent bond between a phosphate group and a hydroxyl moiety of an amino-acid side chain. Because of the size and charge of the phosphate groups, their introduction could have a local, and potentially global, effect on the modified proteins. This effect may translate into modulation of protein activity, subcellular localization, half-life, and ability to interact with other molecules [8,11]. Undoubtedly, the best characterized examples of the molecular effects of phosphorylation on proteins are from high-resolution structural studies (for review [18-20]). For example, some modifications that affect residues that are part of or in the vicinity of catalytic sites and protein docking interfaces may promote or disrupt substrate binding by a combination of steric and electrostatic effects, without apparent major local structural rearrangements Histidine-containing phosphocarrier protein (HPr) [21], isocitrate dehydrogenase [22], signal transducer and activator of transcription [STAT]3B [23], STAT-1 [24], and Stage II sporulation protein (SpoII)AA/SpoIIAB [25]). On the other hand, the modifications could cause conformational changes that result either disorder-to-order transitions (glycogen phosphorylase [26,27]) or increased local flexibility if the native amino-acid packing is disrupted (protein kinase A [28,29], mitogen-activated protein kinase [30], ubiquitin-protein ligase E3 [31], and potassium channel inactivation domain [32]). However, because of technical challenges, few atomic structures of proteins are available in their phosphorylated state. Although atomic models of the proteins in their nonphosphorylated form can provide invaluable clues that may enhance understanding of the molecular impact of modifications on proteins or allow us to predict them [18], no public resource is available that routinely stores and provides this information. Furthermore, current phosphosite databases only address the storage and display of phosphosites [33-35], disregarding the experimental context of the phosphorylation. We have developed the mtcPTM (MitoCheck's post-translational modifications) database to address these needs. The mtcPTM database is a repository of PTMs in human and mouse proteins that aims to preserve and present the experimental evidence that led to the identification of each modification. We show that the graphical display of these data allows intuitive comparisons between phosphorylation patterns from different sources or experiments. The database also contains structural information on those modified protein domains for which the actual structure, or the structure of a close homolog, is available. In addition, we have analyzed in detail this large structural collection to investigate the molecular characteristics of phosphorylatable sites in terms of solvent accessibility, secondary structure preference, and degree of conservation. We report that, in general, modified residues are in flexible/exposed regions and, although they are no more conserved than expected, they present highly variable degrees of conservation. Finally, we elaborate on those cases of phosphorylatable residues that were found buried in the structures, predicting the structural/functional effect of their modification on these proteins. As part of the MitoCheck programme, a European Union-funded project whose overall aim is to study the regulation of mitosis by phosphorylation [36], mtcPTM was originally developed for the study of differential phosphorylation in mitosis. However, its general design is readily applicable to any data, regardless of experimental source. The database is publicly available online, and experimentalists are encouraged to submit their data for storage and display. Results Handling and storage of phosphosite data The mtcPTM database contains data retrieved from literature, protein annotations, and other databases. In the future, the database will also display phosphorylation sites that have been mapped as part of the MitoCheck project. The mtcPTM database therefore handles quite different datasets, for which the available information varies. For example, modifications retrieved from literature and protein annotation are usually recorded as individual residues, in which experimental information can only be recovered by reading the original report. By contrast, high-throughput mass spectrometry (MS) data take the form of phosphorylated positions within peptide sequences. In this case, mtcPTM preserves the experimental context of the phosphosites by grouping the MS peptides into sets according to individual experiments and assigning to each group a hierarchical data structure that summarizes the experimental information. This simple hierarchy comprises data source (for instance, a research group or programme), experimental category (for example, label describing a set of experiments that are undertaken with a combined aim), and individual experiments (data obtained from the same sample). Thus, two experiments undertaken, for example, by MitoCheck to determine the differential phosphorylation state of a protein along the cell cycle would receive the following common labels: 'MitoCheck', 'timing', and a specific label, for example interphase or mitosis. As mentioned above, phosphosites are routinely stored as positions relative to protein sequences [33-35]. However, this has the disadvantage that if the protein entry linked to the phosphosite changes, then the information may be either lost or transferred incorrectly from one database release to the next. By contrast, storage of phosphosites as positions relative to experimentally determined, and thus invariant, peptide sequences allows their automatic update, without information loss, because the peptides can be matched regularly to the most recent version of the corresponding proteome for each new database release. The ability to update and keep track automatically of changes in the data between different releases is important not only to preserve the correct mapping of the phosphosites but also to take full advantage of improvements in genome assemblies and gene builds, especially regarding to the discrimination between splicing variants and handling of promiscuous peptides found in proteins Genome Biology 2007, 8:R90 http://genomebiology.com/2007/8/5/R90 Genome Biology 2007, Volume 8, Issue 5, Article R90 Jiménez et al. R90.3